A recombinant glycine receptor fragment forms homo-oligomers distinct fromits GABA(A) counterpart

The ligand-gated ion channel receptor superfamily includes receptors for gl ycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and ser otonin receptors mediate excitory neurotransmissions, both glycine and GABA A receptors are inhibitory. In this study, a fragment of the human glycine receptor al subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyper-expressed for the first time in Escher ichia coli. This fragment is highly homologous in sequence to the correspon ding fragment of the GABA(A) receptor. The recombinant fragment was found t o have stable beta -rich secondary structure, similar to that found for the homologous GABAA receptor fragment, and ordered tertiary packing, suggesti ng a stable structural domain. Results from laser scattering studies sugges t that the fragment forms trimers in solution. In addition, SDS-induced cha nges in secondary structure were found to occur prior to changes in oligome rization status, suggesting that oligomerization was secondary structure de pendent. A study of quaternary structure using single particle analysis ele ctron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric as sembly of the equivalent GABA, receptor fragment reported by us earlier. Th e fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomeriza tion of the glycine receptor in vivo. Furthermore, the finding that two cou sin receptor fragments form distinct quaternary structures indicates that s equence similarity does not necessarily imply quaternary structure similari ty and, hence, care must be taken when applying a structure model derived f rom studies of individual receptors io the whole ligand-gated ion channel s uperfamily. (C) 2001 Academic Press.